Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase

Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) or nystose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase. The oligosaccharides were identified as 2(2-alpha-D-glucopyranosyl)(m)isokestose; [O-alpha-D-glucopyranosyl-(1-->2)](m)-O-[beta-D-fructofuranosyl-(2-->1)](2)-alpha-D-glucopyranoside: m=1, 2, and 3, and 2(2-alpha-D-glucopyranosyl)(n)nystose; [O-alpha-D-glucopyranosyl-(1-->2)](n)-O-[beta-D-fructofuranosyl-(2-->1)](3)-alpha-D-glucopyranoside: n=1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides. 1H, 13C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC-TOCSY, HMBC, CH(2)-selected E-HSQC, and CH(2)-selected E-HSQC-TOCSY.     

Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. For the example of maltose as the acceptor, if one considers only the linear, unbranched possibilities for alternansucrase, the hypothetical number of potential products increases exponentially as a function of the degree of polymerization (DP). Experimental evidence indicates that far fewer products are actually formed. We show that only certain isomers of DP >4 are formed from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. When the oligosaccharide acceptor products from maltose were separated by size-exclusion chromatography and HPLC, only one pentasaccharide was isolated. Its structure was alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Two hexasaccharides were formed in approximately equal quantities: alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc and alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Just one heptasaccharide was isolated from the reaction mixture, alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. We conclude that the enzyme is incapable of forming two consecutive alpha-(1-->3) linkages, and does not form products with more than two consecutive alpha-(1-->6) linkages. The distribution of products may be kinetically determined.     

Structure of the O-specific polysaccharide chain of the lipopolysaccharide of Bordetella hinzii

The lipopolysaccharide of Bordetella hinzii was analyzed after various chemical degradations by NMR spectroscopy and MALDI mass spectrometry, and the following structure of the polysaccharide chain was determined: 4-O-Me-alpha-GalpNAc3NAcAN-(1-->[-->4)-beta-GlcpNAc3NAcAN-(1-->4)-beta-GlcpNAc3NAcAN-(1-->4)-alpha-GalpNAc3NAcAN-(1-](n)-where GlcNAc3NAcAN and GalNAc3NAcAN stand for 2,3-diacetamido-2,3-dideoxy-glucuronamide and -galacturonamide, respectively. The polysaccharide chain is terminated with a 4-O-methylated GalNAc3NAcAN residue and is rather short (n < or = 5).     

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [--> 3)-beta-D-Gal f -(1 --> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [--> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The mannan cores have also been investigated, and are constituted by a (1 --> 6)-alpha-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 --> 2) linked alpha-mannopyranoses.     

Glucuronic acid directly linked to galacturonic acid in the rhamnogalacturonan backbone of beet pectins.

Sugar-beet pulp was de-esterified and submitted to 72 h hydrolysis by 0.1 M HCl at 80 degrees C. Oligomers containing a single glucuronic acid (GlcA) moiety in addition to n(>/= 2) repeats of the dimer -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1--> were isolated from the hydrolysate by ion-exchange and gel-permeation. Glycosyl linkage composition analysis and 1H NMR studies indicated that the GlcA was attached to O-3 of a galacturonic acid (GalA) residue, as shown for the two pentamers beta-D-GlcpA-(1-->3)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-L-Rhap and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-[beta-D-GlcpA-(1-->3)]-alpha-D-GalpA-(1-->2)-L-Rhap. Substitution by GlcA was estimated as occurring on one GalA residue out of 72 in the rhamnogalacturonan fraction of the backbone of beet pectins.     

Atomic mapping of the interactions between the antiviral agent cyanovirin-N and oligomannosides by saturation-transfer difference NMR

The minimum oligosaccharide structure required for binding to the potent HIV-inactivating protein cyanovirin-N (CV-N) was determined by saturation-transfer difference (STD) NMR spectroscopy. Despite the low molecular mass of the protein (11 kDa), STD-NMR spectroscopy allowed the precise atomic mapping of the interactions between CV-N and various di- and trimannosides, substructures of Man-9, the predominant oligosaccharide on the HIV viral surface glycoprotein gp120. Contacts with mannosides containing the terminal Manalpha(1-->2)Manalpha unit of Man-9 were observed, while (1-->3)- and (1-6)-linked di- and trimannosides showed no interactions, demonstrating that the terminal Manalpha(1-->2)Manalpha structure plays a key role in the interaction. Precise epitope mapping revealed that, for Manalpha(1-->2)ManalphaOMe, Manalpha(1-->2)Manalpha(1-->3)ManalphaOMe, and Manalpha(1-->2)Manalpha(1-->6)ManalphaOMe, the protein is in close contact with H2, H3, and H4 of the nonreducing terminal mannose unit. In contrast, the STD-NMR spectrum of the CV-N/trisaccharide Manalpha(1-->2)Manalpha(1-->2)ManalphaOMe complex was markedly different, with resonances on all sugar units displaying equal enhancements, suggesting that CV-N is able to discriminate between the three structurally related trisaccharides.     

Regioselective synthesis of p-nitrophenyl glycosides of beta-D-galactopyranosyl-disaccharides by transglycosylation with beta-D-galactosidases

The beta-D-galactosidase from porcine liver induced regiospecific transglycosylation of beta-D-galactose from beta-D-Gal-OC6H4NO2-o to OH-6 of, respectively, p-nitrophenyl glycoside acceptors of Gal, GlcNAc and GalNAc to afford beta-Gal-(1-->6)-alpha-Gal-OC6H4NO2-p, beta-Gal-(1--> 6)-beta-Gal-OC6H4NO2-p, beta-Gal-(1-->6)-alpha-GalNAc-OC6H4NO2-p, beta-Gal-(1-->6)-beta-GalNAc-OC6H4NO2-p, beta-Gal-(1-->6)-alpha-GlcNAc-OC6H4NO2-p, and beta-Gal-(1-->6)-beta-GlcNAc-OC6H4NO2-p. The enzyme showed much higher transglycosylation activity for the alpha-glycoside acceptors than the corresponding beta-glycoside acceptors. The regioselectivity of the beta-D-galactosidase from Bacillus circulans ATCC 31382 greatly depended on the nature of the acceptor. When alpha-D-GalNAc-OC6H4NO2-p and alpha-D-GlcNAc-OC6H4NO2-p were used as acceptors, the enzyme showed high potency for regioselective synthesis of beta-Gal-(1-->3)-alpha-GalNAc-OC6H4NO2-p and beta-Gal-(1-->3)-alpha-GlcNAc-OC6H4NO2-p in high respective yields of 75.9 and 79.3% based on the acceptors added. However, replacement of beta-D-Gal-OC6H4NO2-p by beta-D-GalNAc-OC6H4NO2-p did change the direction of galactosylation. The enzyme formed regioselectively beta-Gal-(1-->6)-beta-Gal-OC6H4NO2-p with (beta-Gal-1-->(6-beta-Gal-1-->)n6-beta-Gal-OC6H4NO2-p, n = 1-4). No beta-(1-->3)-linked product was detected during the reaction. Use of the two readily available beta-D-galactosidases facilitates the preparation of (1-->3)- and (1-->6)-linked disaccharide glycosides of beta-D-Gal-GalNAc and beta-D-Gal-GlcNAc.     

Light-harvesting function of carotenoids in photo-synthesis: the roles of the newly found 1(1)Bu- state

This minireview article highlights the energetics and the dynamics of the 1(1)B(u)(-) and 3(1)A(g)(-) states of carotenoids discovered very recently. Those "hidden" covalent states have been revealed by measurements of resonance-Raman excitation profiles of crystalline carotenoids. The dependence of the energies of the low-lying singlet states, including the 1(1)B(u)(+), 3(1)A(g)(-), 1(1)B(u)(-), and 2(1)A(g)(-) states, on the number of conjugated double bonds (n) is in agreement with the extrapolation of those state energies calculated by Tavan and Schulten for shorter polyenes (P. Tavan and K. Schulten, Journal of Chemical Physics, 1986, vol. 85, pp. 6602-6609). It has also been shown that the internal-conversion processes among those singlet states take place in accord with the state ordering, i.e., 1(1)B(u)(+) --> 1(1)B(u)(-) --> 2(1)A(g)(-) --> 1(1)A(g)(-) (the ground state) for carotenoids having n = 9 and 10, whereas 1(1)B(u)(+) --> 3(1)A(g)(-) --> 1(1)B(u) (-) --> 2(1)A(g)(-) --> 1(1)A(g)(-) for carotenoids having n = 11-13. Radiative transitions of 1(1)B(u)(+) --> 2(1)A(g)(-) and 1(1)B(u)(-) --> 2(1)A(g)(-) as well as a branching into the triplet manifold of 1(1)B(u)(-) --> 1(3)A(g) --> 1(3)B(u) have also been found. Those low-lying singlet states of all-trans carotenoids can facilitate multiple channels of singlet-energy transfer to bacteriochlorophyll in the LH2 antenna complexes of purple photosynthetic bacteria. Thus, the newly found 1(1)B(u)(-) and 3(1)A(g)(-) states of carotenoids need to be incorporated into the picture of carotenoid-to-bacteriochlorophyll singlet-energy transfer.     

Differences in the recognition of glucan elicitor signals between rice and soybean: beta-glucan fragments from the rice blast disease fungus Pyricularia oryzae that elicit phytoalexin biosynthesis in suspension-cultured rice cells

Partial acid/enzymatic hydrolysis of the beta-(1-->3, 1-->6)-glucan from the cell walls of the rice blast disease fungus Pyricularia oryzae (Magnaporthe grisea) released elicitor-active fragments that induced phytoalexin biosynthesis in suspension-cultured rice cells. From the digestion of the glucan by an endo-beta-(1-->3)-glucanase, one highly elicitor-active glucopentaose was purified as a reduced compound, tetraglucosyl glucitol. The structure of this tetraglucosyl glucitol as well as two other related tetraglucosyl glucitols was elucidated as follows: (1) Glcbeta(1-->3)Glcbeta(1-->3)(Glcbeta(1-->6)) Glcbeta(1-->3)Glucitol (most active fragment); (2) Glcbeta(1-->3)(Glcbeta(1-->6))Glcbeta(1-->3)Glcbeta (1-->3)Glucitol; and (3) Glcbeta(1-->6) Glcbeta(1-->3)Glcbeta(1-->3)Glcbeta(1-->3)Glucitol. However, a synthetic hexa-beta-glucoside, known as a minimal structural element for the phytoalexin elicitor for soybean cotyledon cells, did not induce phytoalexin biosynthesis in the rice cells. Conversely, the beta-glucan fragment from P. oryzae did not induce phytoalexin biosynthesis in the soybean cotyledon cells, indicating differences in the recognition of glucooligosaccharide elicitor signals in these two plants. Because rice cells have been shown to recognize chitin fragments larger than pentamers as potent elicitors, these results also indicate that the rice cells can recognize at least two types of oligosaccharides from fungal cell walls as signal molecules to initiate defense response.     

Application of MS and NMR to the structure elucidation of complex sugar moieties of natural products: exemplified by the steroidal saponin from Yucca filamentosa L

An approach, using well characterized procedures, is presented that should be of general applicability for the structural elucidation of complex sugar moieties of natural products. The methods used are exemplified by the structure elucidation of a new gitogenin-based steroidal saponin that has a strong leishmanicidal activity similar to preparations used in clinical practice and has been isolated by bioactivity-guided fractionation of the ethanolic extract of Yucca filamentosa L. leaves. The saponin has been characterized as 3-O-((beta-D-glucopyranosyl-(1-->3)- beta-D-glucopyranosy-(1-->2))(alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3))-beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranosyl)-25R,5alpha-spirostan-2alpha,3beta-diol.     

Mitogen-induced rapid phosphorylation of serine 795 of the retinoblastoma gene product in vascular smooth muscle cells involves ERK activation

We examined the relationship between mitogen-activated MEK (mitogen and extracellular signal-regulated protein kinase kinase) and phosphorylation of the gene product encoded by retinoblastoma (hereafter referred to as Rb) in vascular smooth muscle cells. Brief treatment of the cells with 100 nm angiotensin II or 1 microm serotonin resulted in serine phosphorylation of Rb that was equal in magnitude to that induced by treating cells for 20 h with 10% fetal bovine serum ( approximately 3 x basal). There was no detectable rapid phosphorylation of two close cousins of Rb, p107 and p130. Phosphorylation state-specific antisera demonstrated that the rapid phosphorylation occurred on Ser(795), but not on Ser(249), Thr(252), Thr(373), Ser(780), Ser(807), or Ser(811). Phosphorylation of Rb Ser(795) peaked at 10 min, lagging behind phosphorylation of MEK and ERK (extracellular signal-regulated protein kinase). Rb Ser(795) phosphorylation could be blocked by PD98059, a MEK inhibitor, and greatly attenuated by apigenin, an inhibitor of the Ras --> Raf --> MEK --> ERK pathway. The effect also appears to be mediated by CDK4. Immunoprecipitation/immunoblot studies revealed that serotonin and angiotensin II induced complex formation between CDK4, cyclin D1, and phosphorylated ERK. These studies show a rapid, novel, and selective phosphorylation of Rb Ser(795) by mitogens and demonstrate an unexpected rapid linkage between the actions of the Ras --> Raf --> MEK --> ERK pathway and the phosphorylation state of Rb.     

Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.

The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.     

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).     

Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthes japonica.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The multiple activities of polyphosphate kinase of Escherichia coli and their subunit structure determined by radiation target analysis

Polyphosphate kinase (PPK), the principal enzyme required for the synthesis of inorganic polyphosphate (polyP) from ATP, also exhibits other enzymatic activities, which differ significantly in their biochemical optima and responses to chemical agents. These several activities include: polyP synthesis (forward reaction), nATP --> polyP(n) + nADP (Equation 1); ATP synthesis from polyP (reverse reaction), ADP + polyP(n) --> ATP + polyP(n - 1) (Equation 2); general nucleoside-diphosphate kinase, GDP + polyP(n) --> GTP + polyP(n - 1) (Equation 3); linear guanosine 5'-tetraphosphate (ppppG) synthesis, GDP + polyP(n) --> ppppG + polyP(n - 2) (Equation 4); and autophosphorylation, PPK + ATP --> PPK-P + ADP (Equation 5). The Mg(2+) optima are 5, 2, 1, and 0.2 mM, respectively, for the activities in Equations 1, 2, 3, and 4. Inorganic pyrophosphate inhibits the activities in Equations 1 and 3 but stimulates that in Equation 4. The kinetics of the activities in Equations 1, 2, and 3 are highly processive, whereas the transfer of a pyrophosphoryl group from polyP to GDP (Equation 4) is distributive and demonstrates a rapid equilibrium, random Bi-Bi catalytic mechanism. Radiation target analysis revealed that the principal functional unit of the homotetrameric PPK is a dimer. Exceptions are a trimer for the synthesis of ppppG (Equation 4) and a tetrameric state for the autophosphorylation of PPK (Equation 5) at low ATP concentrations. Thus, the diverse functions of this enzyme involve different subunit organizations and conformations. The highly conserved homology of PPK among 18 microorganisms was used to determine important residues and conserved regions by alanine substitution, by site-directed mutagenesis, and by deletion mutagenesis. Of 46 single-site mutants, seven exhibit none of the five enzymatic activities; in one mutant, ATP synthesis from polyP is reduced relative to GTP synthesis. Among deletion mutants, some lost all five PPK activities, but others retained partial activity for some reactions but not for others.     

Polaprezinc prevents ongoing thioacetamide-induced liver fibrosis in rats

AimsCirrhotic patients commonly have a liver zinc deficiency, which may aggravate liver fibrosis due to the lack of antioxidative effects of zinc. This study examined the ability of polaprezinc, N-(3-aminopropionyl)-l-histidinato zinc, to prevent fibrosis in a rat model of thioacetamide (TAA)-induced hepatic fibrosis.Main methodsLiver cirrhosis was induced by orally administering TAA for 20 weeks. The rats were cotreated with one of the following for the last 10 weeks of TAA treatment: (1) polaprezinc (50 or 200 mg/kg/day); (2) l-carnosine (155 mg/kg/day), which contained equal amounts of l-carnosine as 200 mg/kg/day polaprezinc; (3) zinc sulfate (112 mg/kg/day) or (4) zinc-l-aspartic complex (317.8 mg/kg/day). Both zinc supplementations contained equal amounts of zinc as high-dose polaprezinc.Key findingsHepatic zinc levels fell significantly in rats treated with TAA for 20 weeks. Cotreating with high-dose polaprezinc and zinc-l-aspartic complex for 10 weeks prevented hepatic zinc loss. Hepatic hydroxyproline and tissue inhibitor of metalloproteinases-1 (TIMP-1) were significantly higher in rats treated with TAA for 20 weeks than 10 weeks, whereas polaprezinc prevented changes in these fibrosis markers and reduced hepatic transforming growth factor-β1 protein concentration, macroscopic and histologic changes. TAA caused oxidative stress-related changes in the liver that were prevented by high-dose polaprezinc and partially by zinc-l-aspartic complex. Treatment with l-carnosine, low-dose polaprezinc or zinc sulfate for 10 weeks did not affect liver fibrosis progression or oxidative stress-related changes.SignificancePolaprezinc may prevent ongoing fibrosis by preventing zinc depletion, oxidative stress and fibrosis markers in cirrhotic livers.     

Correlation between substitutions in penicillin-binding protein 1 and amoxicillin resistance in Helicobacter pylori

The correlation between the substitutions of penicillin-binding protein 1 (PBP1) and amoxicillin resistance was studied for the determination of the substitutions in PBP1 which confer amoxicillin resistance in Helicobacter pylori. By the comparison of the amino acid sequences of PBP1 in the amoxicillinresistant (n=3), low-susceptible (n=3), and susceptible (n=13) H. pylori isolates, the substitution Asn562-->Tyr, which is adjacent to KTG motif (555-557), was common and specific to amoxicillin-resistant H. pylori. Additionally, all amoxicillin-resistant isolates had multiple substitutions such as Ser414-->Arg in the transpeptidase region of PBP1 of H. pylori. Furthermore all transformants obtained by the natural transformation using the pbp1 genes of amoxicillin-resistant H. pylori isolates had multiple substitutions including Asn562-->Tyr. These results suggest that multiple amino acid substitutions in the transpeptidase region of PBP1 are closely related to amoxicillin resistance in H. pylori.     

Clostridium difficile cell-surface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units

Clostridium difficile is a Gram-positive bacterium that is known to be a cause of enteric diseases in humans. It is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. Recently, large outbreaks of C. difficile-associated diarrhea have been reported internationally, and there have been reports of increases in severe disease, mortality and relapse rates. At the moment, there is no vaccine against C. difficile, and the medical prevention of C. difficile infection is mostly based on the prophylactic use of antibiotics; however, this has led to an increase in the incidence of the disease. Here, we describe the chemical structure of C. difficile cell-surface polysaccharides. The polysaccharides of three C. difficile strains were structurally analyzed; ribotype 027 (North American pulsotype 1) strain was observed to express two polysaccharides, one was composed of a branched pentaglycosyl phosphate repeating unit: [-->4)-alpha-l-Rhap-(1-->3)-beta-D-Glcp-(1-->4)-[alpha-l-Rhap-(1-->3]-alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->P] and the other was composed of a hexaglycosyl phosphate repeating unit: [-->6)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->4)-alpha-D-Glcp-(1-->4)-[beta-D-Glcp-(1-->]-beta-D-GalpNAc-(1-->3)-alpha-D-Manp-(1-->P]. The latter polysaccharide was also observed to be produced by strains MOH900 and MOH718. The results described here represent the first literature report describing the covalent chemical structures of C. difficile cell-surface polysaccharides, of which PS-II appears to be a regular C. difficile antigen. These C. difficile teichoic-acid-like polysaccharides will be tested as immunogens in vaccine preparations in a rat and horse model.     

alpha-D-Glcp-(1<-->1)-beta-D-Galp-containing oligosaccharides, novel products from lactose by the action of beta-galactosidase

A mixture of oligosaccharides produced by beta-galactosidase using lactose as a substrate was fractionated according to degree of polymerization using gel filtration, followed by high-pH anion-exchange chromatography. The fractions obtained were analyzed using monosaccharide analysis, methylation analysis, mass spectrometry, and NMR spectroscopy. Twelve novel non-reducing oligosaccharides were characterized, namely, [beta-D-Galp-(1-->4)]n-alpha-D-Glcp- (1<-->1)-beta-D-Galp[-(4<--1)-beta-D-Galp]m, with n, m = (1, 2, 3, or 4) and beta-D-Galp-(1-->2)-alpha-D-Glcp- (1<-->1)-beta-D-Galp.     

Chromene-3-carboxamide derivatives discovered from virtual screening as potent inhibitors of the tumour maker,AKR1B10

A human aldose reductase-like protein, AKR1B10 in the aldo-keto reductase (AKR) superfamily, was recently identified as a therapeutic target in the treatment of several types of cancer. In order to identify potential leads for new inhibitors of AKR1B10, we adopted the virtual screening approach using the automated program icm, which resulted in the discovery of several chromene-3-carboxamide derivatives as potent competitive inhibitors. The most potent (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide inhibited the reductase activity of AKR1B10 with a K_i value of 2.7 nM, and the metabolism of farnesal and 4-hydroxynonenal in the AKR1B10-overexpressed cells from 0.1 μM with an IC₅₀ value equal to 0.8 μM.